Process for the chemical synthesis of oligonucleotides

ABSTRACT

Use of a dansylethoxycarbonyl group as base-labile 5&#39;-hydroxyl protective group in the chemical synthesis of DNA and RNA and suitable synthetic processes. 
     The ease of detection of the dansyl protective group and the ease of elimination from the sugar residue of the nucleotide without side reactions make oligonucleotide synthesis possible with high yields in very small quantities.

This is a division of application Ser. No. 08/437,566, filed May 9,1995, now U.S. Pat. No. 5,631,362, which is a continuation of Ser. No.08/219,239 filed Mar. 28, 1994, now abandoned, which is a continuationof Ser. No. 07/757,924 filed Sep. 12, 1991, abandoned.

The chemical polycondensation of mononucleotides is an important methodfor the preparation of deoxyribonucleic acid (DNA) or ribonucleic acid(RNA).

A fundamental problem in the chemical synthesis of DNA or RNA is to findsuitable protective groups for the amino and hydroxyl groups on thenucleoside bases and the sugar residues. These protective groups must,on the one hand, be stable under the conditions of the polycondensationreaction, i.e. during the formation of the phosphodiester linkage, andmust, on the other hand, be sufficiently labile for it to be possible toremove them again at the end of the reaction without cleaving thephosphodiester linkage (H. G. Khorana; Pure Appl. Chem. 17 (1968) 349).

The chemical synthesis of RNA is particularly difficult because theribose sugar residue carries two hydroxyl groups, both of which must beprotected. Moreover, before each polycondensation step, the protectivegroup on the 5'-hydroxyl group must be eliminated again selectively,i.e. without eliminating the 2'-hydroxyl protective group. On the otherhand, the protective group on the 2'-hydroxyl group must be eliminatedonly at the end of the RNA synthesis, specifically under conditionswhich do not lead to any cleavage or isomerization of the phosphodiesterlinkages (C. B. Reese, Nucleic Acids and Molecular Biology, Vol 3 (F.Eckstein & D. M. J. Lilley eds.) Springer-Verlag, Weinheim).

One possibility for the selective elimination of the 5'-hydroxylprotective group without eliminating the 2'-hydroxyl protective group isachievable by combining a base-labile 5'-hydroxyl protective group withan acid-labile 2'-hydroxyl protective group (Chr. Lehmann et al. (1989)Nucleic Acids Res. 17, 2379-2390, No. 7). The use of a base-labile5'-hydroxyl protective group is also advantageous in the synthesis ofDNA because there is generally no cleavage under the mild non-acidhydrolysis conditions of the phosphodi- and -triester linkages alreadyformed in the synthesis. In addition, depurination of the nucleotides asdescribed by E. Sonveaux (E. Sonveaux (1986), Bioorganic Chemistry 14,286) is avoided under the mild non-acid hydrolysis conditions. Anotherrequirement to be met by the 5'-hydroxyl protective group in DNA as wellas in RNA synthesis is that the protective group be detectable easilyand highly sensitively. It is possible in this way to follow especiallywell the degree of conversion in the individual reaction steps and toachieve conversion which is as complete as possible. This makes itpossible to prepare especially long oligonucleotides in high yield. Thisalso permits small synthesis mixtures in the nanomolar to picomolarrange to be carried out.

It has now been found, surprisingly, that the dansylethoxycarbonyl group(Dans-EOC) can be employed as base-labile 5'-hydroxyl protective groupin chemical oligonucleotide synthesis.

The invention therefore relates to

1. a process for the preparation of the compound of the formula (I)##STR1## which comprises reacting the compound of the formula (II)##STR2## with a chlorocarbonyl donor. 2. A compound of the formula(IIIa) or (IIIb) ##STR3## in which R₁ is hydrogen or, independently ofone another, a group of the formula ##STR4## and B is ##STR5## with R₂being, in each case independently of one another, a group of the formula##STR6## or B is ##STR7## with R₃ being hydrogen or ##STR8## and R₄being ##STR9## or B is ##STR10## with R₅ being --OH, ##STR11## Y=H,alkyl (C₁ -C₄), especially CH₃, ##STR12## 3. A process for thepreparation of a compound of the formula (IIIa) or (IIIb) by reactingthe compound of the formula (I) with an appropriate compound of theformula (IVa) or (IVb) ##STR13## in which R₁ and B have the abovemeaning, in the presence of a base, preferably pyridine or a mixturecomposed of tetrahydrofuran, dioxane, methylene chloride, chloroformand/or acetonitrile and of a compound of the formula

    NR.sub.20 R.sub.21 R.sub.22

in which R₂₀, R₂₁ and R₂₂ are, identically or independently differently,hydrogen or the C₁ -C₄ -alkyl group, preferably a trimethyl, triethyl ordiisopropyl group.

4. A compound of the formula (Va) or (Vb) ##STR14## in which DansEOC, R₁and B have the abovementioned meaning, and R₆ and R₇ are, identically orindependently differently, a C₁ -C₈ -alkyl, preferably an isopropyl orC₅ -C₁₂ -cycloalkyl group, preferably up to C₈, benzyl or phenyl or,together with the nitrogen atom to which they are bonded, a saturated orunsaturated heterocyclic ring which can optionally contain furtherhetero atoms and substituents and R₈ is group of the formula ##STR15##or a benzyl group which is unsubstituted or ring-substituted one or moretimes, preferably unsubstituted, where the substituent(s) is,independently of one another, a halogen, a C₁ -C₄ -alkyl, nitro, methoxyor carboxyl group.

5. A process for the preparation of a compound of the formula (Va) or(Vb) by reacting the compound of the formula (IIIa) or (IIIb) with acompound of the formula (VI) ##STR16## in which R₆, R₇ and R₈ have theabove meaning, and Z is chlorine or bromine or a radical of the formula--NR₉ R₁₀ where the same radicals as for R₆ are suitable for R₉ and R₁₀,independently of one another, when Z equals chlorine, in the presence ofa base, preferably pyridine or a mixture of tetrahydrofuran, dioxane,methylene chloride, chloroform and/or acetonitrile with a C₁ -C₄-trialkylamine, preferably a trimethyl-, triethyl- or diisopropylethylamine, or when Z is a radical of the formula --NR₉ R₁₀, then in thepresence of a compound of the formula HNR₁₁ R₁₂ R₁₃ !.sup.(+) X.sup.(-)where R₁₁, R₁₂ and R₁₃ are, identically or independently differently, aC₁ -C₄ -alkyl group and X is halogen, especially chlorine, or tetrazole,preferably in the presence of tetrazole.

6. A process for the preparation of oligonucleotides from compounds ofthe formula (Va) and/or (Vb), which comprises a compound of the formula(Va) or (Vb)

1. being reacted with a compound of the formula (VIIa) or (VIIb)##STR17## in which B and R₁ have the abovementioned meaning, and G hasthe same meaning as R₁ or is a polymeric support which is bonded via the2'-hydroxyl or 3'-hydroxyl group of the compound of the formula (VIIa)or (VIIb),

2. the resulting compounds being oxidized

3. the dansylethoxycarbonyl group being eliminated,

4. the resulting compound being reacted with a compound of the formula(Va) or (Vb) and

5. reaction steps 2-4 being repeated up to the required chain length.

7. A compound of the formula (VIIIa) or (VIIIb) ##STR18## in whichDansEOC, R₁ and B have the abovementioned meaning, and K.sup.(+) is acation, especially HN(C₂ H₅)₃ !.sup.(+).

8. A process for the preparation of a compound of the formula (VIIIa) or(VIIIb) by reacting a compound of the formula (IIIa) or (IIIb) with acompound of the formula (IX)

    PR.sub.14 R.sub.15 R.sub.16                                (IX)

in which R₁₄, R₁₅ and R₁₆ are, identically or independently differently,hydrogen or a C₁ -C₈ -alkyl, C₁ -C₈ -fluoroalkyl or aryl group,preferably a 2,2,2-trifluoroethyl, 1,1,1,3,3,3,-hexafluoro-2-propyl,ethyl or phenyl group in the presence of a base.

9. A process for the preparation of a compound of the formula (VIIIa) or(VIIIb) by reacting a compound of the formula (IIIa) or (IIIb) with acompound of the formula (X)

    PR.sub.17 R.sub.18 R.sub.19                                (X)

in which R₁₇, R₁₈, and R₁₉ are, identically or independentlydifferently, chlorine, bromine or a C₁ -C₈ -alkylamino or1,2,4-triazolyl group, preferably a 1,2,4-triazolyl group, in thepresence of a base with subsequent hydrolysis.

10. A process for the preparation of oligonucleotides from compounds ofthe formula (VIIIa) and/or (VIIIb), which comprises a compound of theformula (VIIIa) or (VIIIb)

1. being reacted with a compound of the formula (VIIa) or (VIIb),

2. the dansylethoxycarbonyl group being eliminated,

3. the resulting compound being reacted with a compound of the formula(VIIIa) or (VIIIb),

4. reaction steps 2 and 3 being repeated up to the required chain lengthand

5. the resulting oligonucleotide being oxidized.

To introduce the dansylethoxycarbonyl group into the nucleoside,2-dansylethyl chloroformate hydrochlorid was reacted with a nucleosidein which, depending on the nucleoside, the amino and hydroxyl groups inthe nucleoside base are protected by suitable groups. Examples ofsuitable protective groups for the 6-amino group of adenine are thet-butyloxycarbonyl, benzoyl, 4-(t-butyl)benzoyl orpara-nitrophenylethyloxycarbonyl group, especially the benzoyl or thepara-nitrophenylethyloxycarbonyl group.

Examples suitable for the 2-amino group of guanine are the isobutyryl,4-(t-butyl)phenylacetyl or para-nitrophenylethyloxycarbonyl group,especially the isobutyryl or the para-nitrophenylethyloxycarbonyl group.The 6-hydroxyl group of guanine and the 4-hydroxyl group of uracileither generally remain unprotected or are protected by apara-nitrophenylethyl group. In the case of cytosine, the 4-amino groupis protected, for example, by a benzoyl, 4-(t-butyl)benzoyl orpara-nitrophenylethyloxycarbonyl group, especially the benzoyl or thepara-nitrophenylethyloxycarbonyl group. Thymidine generally remainsunprotected. The 3-N group in uridine is protedted, for example, by aBoc or an amisoyl group.

It is also possible in place of the natural nucleoside bases to usemodified nucleoside bases whose amino or hydroxyl groups can beprotected in an analogous manner by the abovementioned protectivegroups. Examples of nucleosides with modified bases are inosine,8-aza-7-de-azaadenosine, tubercidin, nebularine, xanthosine,2-aminoadenosine or pyridopyrimidine nucleosides. The nucleosides can bebought, and the introduction of the individual protective groups can becarried out, for example, by the method C. Lehmann et al. (1989), C. B.Reese (1989) "The Chemical Synthesis of Oligo and Polyribonucleotides"in Nucleic Acids and Molecular Biology 3, F. Eckstein & D. M. J. Lilley(eds.), Springer Verlag Berlin, Heidelberg!, E. Sonveaux (1986),Bioorganic Chemistry 14, 274-325 or E. Uhlmann and A. Peyman (1990),Chemical Reviews 90, 543-584, No. 4.

When ribonucleotides are used it is necessary also to protect the2'-hydroxyl group of the ribose residue, in addition to the hydroxyl andamino group of the nucleotide bases. As already mentioned, it isimportant for RNA synthesis to be able, by the choice of a suitablecombination of 5'-hydroxyl and 2'-hydroxyl protective group, to removethe 5'-hydroxyl protective group selectively, i.e. without eliminationof the 2'-hydroxyl protective group.

It is now possible to eliminate selectively the dansylethoxycarbonylgroup as 5'-hydroxyl protective group under non-acid conditions in thepresence of acid-labile 2'-hydroxyl protective groups. Examples ofacid-labile 2'-hydroxyl protective groups which can be used are the4-methoxy-4-tetrahydropyranyl, tetrahydropyranyl, t-butyldimethylsilyl,2-nitrobenzyl, 1-(2-chloro-4-methylphenyl)-4-methoxy-4-piperidinyl, orthe 1-(2-fluorophenyl)-4-methoxy-4-piperidinyl group. Thedansylethoxycarbonyl group is preferably eliminated in an aprotic polarsolvent, especially acetonitrile or pyridine having 1 to 3, preferably1.5 to 2.5 mole equivalents of DBU (=1,5-diazabicyclo5.4.0!undec-5-ene). It is possible as an alternative to employ basessuch as TMG (=N¹,N¹,N²,N² tetramethylguanidine) or C₁ -C₄-trialkylamines such as, triethylamine for the elimination.

2-Dansylethyl chloroformate hyrochloride was prepared as startingcompound for the 5'-hydroxyl protective group of the ribose ordeoxyribose residue by reacting 2-dansylethanol with a chlorocarbonyldonor such as, for example, trichloromethyl chloroformate, (diphosgene)and/or phosgene, preferably trichloromethyl chloroformate in thepresence of a polar, aprotic solvent. In a preferred embodiment, thereaction was carried out in the presence of a single polar, aproticsolvent, especially in the presence of acetonitrile. The molar ratio of2-dansylethanol to the chlorocarbonyl donor was 0.5-1 to 1-2, preferably1 to 1-2, in particular 1 to 1.5-2. The reaction temperature was in arange from -20° C. to the boiling point of the reaction mixture,preferably from -5° C. to +20° C., in particular from 0° C. to 5° C.

The process according to the invention results in 2-dansylethylchloroformate hydrochloride as a pure product whose composition wasconfirmed by elemental analysis. This is so surprising because A.Takadate et al. (A. Takadate et al. (1983) Yakugaku Zasshi 103, 982-966)reacted 2-dansylethanol with trichloromethyl chloroformate to give aproduct whose melting point is about 20° C. lower than the productsynthesized in the said process. 2-Dansylethanol can be prepared, forexample, by the method of S. Goya et al. (S. Goya et al (1981) YakugakuZasshi 101, 1164).

The reaction of 2-dansylethyl chloroformate hydrochloride with theprotected nucleoside can be carried out, for example, in analogy to thereaction with 9-fluorenylmethoxycarbonyl chloride by the method of C.Lehmann et al. (1989) in the presence of a base. Suitable as base areorganic bases, especially pyridine or a mixture composed oftetrahydrofuran, dioxane, methylene chloride, chloroform and/oracetonitrile and of a compound of the formula

    NR.sub.20 R.sub.21 R.sub.22

in which R₂₀, R₂₁ and R₂₂ are, identically or independently differently,hydrogen or a C₁ -C₄ -alkyl, preferably a trimethyl, triethyl ordiisopropyl group. If the substrate used therefore is a 2'-protectedribonucleoside, the result is a mixture of products composed of thedansylethyloxycarbonyl-ribonucleoside and thebis-dansylethyloxycarbonyl-ribonucleoside as by-product. This mixture ofproducts can be employed directly in the subsequent phosphorylationreaction. It is also possible, and preferable, for the mixture to bepurified by, for example, flash chromatography. Thebis-dansylethyloxycarbonyl-ribonucleoside which has been removed canthen subsequently be cleaved, for example with DBU, into the dansyl-freeribonucleoside which in turn can be used as starting compound for thedansylation reaction.

To synthesize 2',5'-linked oligoribonucleotides which, for example, astri- or tetraadenylate inhibit protein biosynthesis (Kerr, I. M. &Brown, R. E. (1978) Proc. Natl. Acad. Sci. USA. 75, 256-260), it ispossible to react a nucleoside which is protected in accordance with theabove description and has a free 2'-hydroxyl group with 2-dansylethylchloroformate hydrochloride in an analogous manner.

The dansylated nucleoside with the 2'- or 3'-hydroxyl group still freeon the sugar residue is generally phosphitylated. It is possible to useas phosphitylation reagent, for example, a compound of the formula (VI)##STR19## in which R₆ and R₇ are, identically or independentlydifferently, C₁ -C₈ -alkyl, preferably an isopropyl or C₅ -C₁₂-cycloalkyl group, preferably up to C₈, a benzyl or a phenyl group, or,together with the nitrogen atom to which they are bonded, a saturated orunsaturated heterocyclic ring which can optionally contain furtherhetero atoms and substituents

R₈ is a group of the formula ##STR20## or CH₃, or a benzyl group whichis unsubstituted or ring-substituted one or more times, preferablyunsubstituted, where the substituent(s) is, independently of oneanother, for example a halogen, a C₁ -C₄ -alkyl, nitro, methoxy orcarboxyl group,

z is chlorine, bromine or a radical of the formula --NR₉ R₁₀ where R₉and R₁₀ are, identically or independently differently, a C₁ -C₈ -alkyl,preferably an isopropyl or C₅ -C₁₂ -cycloalkyl group, preferably up toC₈, a benzyl or a phenyl group.

Preferably used as phosphitylation reagent was a compound of the formula(VII) with Z=chlorine, R₆ and R₇ each an isopropyl radical and R₈ equalsa group of the formula ##STR21## The reaction was generally carried outin an organic solvent such as tetrahydrofuran or methylene chloride,preferably methylene chloride, in the presence of 1 to 8, preferably 1to 6, in particular 1 to 4, mole equivalents of an organic base such aspyridine or a mixture of tetrahydrofuran (THF), dioxane, methylenechloride, chloroform and/or acetonitrile and of a C₁ -C₄ -trialkylamine,preferably a trimethyl-, triethyl- or diisopropylethyl-amine, especiallydiisopropylethyl-amine. If Z is a radical of the formula --NR₉ R₁₀, thereaction was preferably carried out in the presence of a compound of theformula (HNR₁₁ R₁₂ R₁₃).sup.(+) X.sup.(-) where R₁₁, R₁₂ and R₁₃ are,identically or independently differently, a C₁ -C₄ -alkyl group andX.sup.(-) =halide, especially a chloride, or a tetrazolide, or atetrazole, preferably in the presence of tetrazole. The molar ratio ofdansylated nucleoside to phosphitylation reagent was 1 to 1-4,preferably 1 to 2-4, in particular 1 to 2.5-3.5.

The compounds of the formula (Va) or (Vb) obtained in this way cansubsequently be employed for oligonucleotide synthesis. In this, thesugar residues of the nucleotides are deoxyribose for DNA synthesis andribose for RNA synthesis, but mixtures of deoxyribose and ribose for thesynthesis of an oligonucleotide composed of regularly or irregularlyarranged deoxyribose and ribose sugar residues are also possible.Furthermore, the oligonucleotide can have a regular or irregularstructure composed of mononucleotides of the formula (Va) and (Vb). Theoligo- and polynucleotide synthesis can be carried out in a manneranalogous to the phosphoramidite method as described, for example, byChr. Lehmann et al. (1989).

There are in principle two possibilities for the synthesis ofoligonucleotides. On the one hand, the synthesis can take place insolution, for example by the method described by C. B. Reese (C. B.Reese (1989) "The Chemical Synthesis of Oligo- and Poly-ribonucleotides"in Nucleic Acids and Molecular Biology (F. Eckstein & D. M. J. Lilley,eds.) 3, 164-181).

On the other hand, the oligonucleotide synthesis can take place on thesolid phase, for example on nucleoside-functionalized glass, (K. P.Stengele & W. Pfleiderer (1989) Nucleic Acids Res. Symp. Ser. 21, 101,K. P. Stengele & W. Pfleiderer (1990) Tetrahedron Lett. 31, 2549 or Chr.Lehmann et al. (1989) Nucleic Acids Res. 17, 2379-2390, No. 7). Ingeneral, the solid-phase synthesis is the preferred method.

For this, the following reaction sequence was preferably chosen:

1. Reaction of a compound of the formula (Va) or (Vb) with thenucleoside of the formula (VIIa) or (VIIb) ##STR22## in which B and R₁have the abovementioned meaning, and G has the same meaning as R₁ or isa polymeric support which is bonded via the 2'-hydroxyl or 3'-hydroxylgroup of the compound of the formula (VIIa) or (VIIb), in the presenceof a weak acid, for example tetrazole or p-nitrophenyltetrazole.

2. Trapping of unreacted compounds of the formula (VIIa) or (VIIb), forexample with acetic anhydride.

3. Oxidation to phosphate, phosphoramidate or to the thiophosphate, forexample with iodine, sulfur or iodine/amine.

4. Elimination of the dansylethoxycarbonyl group, for example with DBUin acetonitrile.

5. Reaction of the resulting support-linked compound with a compound ofthe formula (Va) or (Vb).

6. Repetition of reaction steps 2 to 6 to give the required chain lengthof the oligonucleotide.

The compounds of the formula (Va) or (Vb) and (VIIa) or (VIIb) werepreferably reacted at -20 to +100° C., in particular at room temperaturein the presence of, for example, tetrazole or para-nitrophenyltetrazoleas weak acids. The oxidation was carried out at a temperature of -80 to100° C., preferably at -20 to +60° C., in the presence of iodine, sulfuror iodine in the presence of an amine (A. Jager et al. Biochemistry 27,7237 (1988)). When a mixture of iodine, water and an organic base suchas lutidine or pyridine was used, the oxidation was preferably carriedout at room temperature. On the other hand, when a mixture of elementalsulfur, toluene and an organic base was used, the oxidation waspreferably carried out at 60° C.

The synthesized oligonucleotides were generally composed of 2 to about200, preferably 2 to 100, in particular 2 to 20 mononucleotides.

As an alternative to the phosphitylation, the dansylated nucleoside withthe 2'- or 3'-hydroxyl group still free on the sugar residue can also beconverted into the H-phosphonate of the formula (VIIIa) or (VIIIb).##STR23## in which DansEOC, R₁ and B have the abovementioned meaning,and K.sup.(+) is a cation, especially NH(C₂ H₅)₃ !.sup.(+) (R.Stromberg, Chem. Commun. 1987, 1; B. C. Froehler, P. G. Ng, M. D.Matteucci, Nucleic Acids Res. 14 (1986) 5399; M. Tahalu et al., Chem.Lett. 1988, 1675).

This generally entails a compound of the formula (IIIa) or (IIIb) beingreacted with a compound of the formula (IX) or (X)

    PR.sub.14 R.sub.15 R.sub.16                                (IX)

    PR.sub.17 R.sub.18 R.sub.19                                (X)

in which R₁₄, R₁₅ and R₁₆ are, identically or independently differently,hydrogen or a C₁ -C₈ -alkyl, C₁ -C₈ -fluoroalkyl or aryl group and R₁₇,R₁₈ and R₁₉ are, identically or independently differently, chlorine,bromine or a C₁ -C₈ -alkylamino or a 1,2,4-triazolyl group.

The dansylated nucleoside was preferably reacted withbis(2,2,2-trifluoroethyl) H-phosphonate,bis(1,1,1,3,3,3-hexafluoro-2-propyl) H-phosphonate, triethyl phosphite,triphenyl phosphite or with PCl₃, tri(dialkylamino)phosphines ortris(1,2,4-triazolyl) phosphite, preferably tris(1,2,4-triazolyl)phosphite, particularly preferably with PCl₃ after activation withimidazole/triethylamine with subsequent hydrolysis to the H-phosphonate.

The reaction was carried out in an organic solvent such astetrahydrofuran or methylene chloride, preferably methylene chloride, inthe presence of 1-50 mole equivalents, preferably 10-50, in particular30-50 mole equivalents of an organic base such as C₁ -C₄ -trialkylamineor N-C₁ -C₄ -alkylmorpholine, preferably N-methylmorpholine. The molarratio of dansylated nucleoside to the phosphonylation reagent was 1 to1-10, preferably 1 to 2-8, in particular 1 to 5.

The compounds of the formula (VIIIa) or (VIIIb) obtained in this way cansubsequently be employed for the oligonucleotide synthesis. For this,the sugar residues of the nucleotides are deoxyribose for the DNAsynthesis and ribose for RNA synthesis, but mixtures of deoxyribose andribose can also be used for synthesizing an oligonucleotide composed ofregularly or irregularly arranged deoxyribose and ribose sugar residues.Furthermore, the oligonucleotide can have a regular or irregularstructure composed of mononucleotides of the formula (VIIIa) and(VIIIb).

The oligonucleotide synthesis can be carried out by the H-phosphonatemethod as described, for example, by B. C. Froehler et al. (Froehler, B.C. (1986) Nucleic Acids Res. 14, 5399-5407, No. 13), in which case theacid elimination of the 5'-hydroxyl protective group is replaced by thebasic elimination of the dansylethoxycarbonyl group.

The synthesis can in principle be carried out either in solution, forexample in analogy to the method of C. B. Reese (1989), or on the solidphase, for example in analogy to the method of B. C. Froehler (1986).The solid-phase synthesis is generally preferred.

The following reaction sequence was preferably chosen for this:

1. Reaction of a compound of the formula (VIIIa) or (VIIIb) with thenucleoside-bonded polymeric support of the formula (VIIa) or (VIIb) inthe presence of an acid chloride, for example pivaloyl chloride oradamantoyl chloride

2. Elimination of the dansylethoxycarbonyl group, for example with DBU

3. Reaction of the resulting compound with a compound of the formula(VIIIa) or (VIIIb)

4. Repetition of step 2 and 3 up to the required chain length of theoligonucleotide

5. Oxidation, to the phosphate, phosphoramidate or to the thiophosphate,for example with iodine or sulfur or amine/CCl₄ /triphenylphosphine(Jager et al. see above).

The compounds of the formula (VIIIa) or (VIIIb) and (VIIa) and (VIIb)were preferably reacted at a temperature of -20° C. to +100° C., inparticular at room temperature in the presence of, for example, pivaloylchloride as acid. The oxidation was carried out, for example, withiodine in a solvent mixture composed in general of pyridine,N-methylimidazole, water, THF at room temperature.

The oligonucleotides synthesized by the H-phosphonate method describedwere generally composed of 2 to about 200, preferably 2 to 110, inparticular 2 to 40 mononucleotides.

The advantages of oligonucleotide synthesis from dansylatedmononucleotides by the phosphoramidite or by the H-phosphonate methodare

a) the dansyl group is easy to detect because of its strong fluorescenceat 550 nm,

b) synthesis of poly- and oligonucleotides down to the picomole range,

c) removal of the 5'-hydroxyl dansyl protective group withoutelimination of other hydroxyl protective groups on the nucleotide baseor on the sugar residue

d) synthesis of oligoribonucleotides and oligodeoxyribonucleotides,especially of oligoribonucleotides,

e) solid-phase synthesis of oligonucleotides in high yields and in largechain lengths.

Another advantage of the use of the 5'-hydroxyl dansyl protective groupin RNA synthesis is that the 2'-hydroxyl groups of the ribose residuecan remain protected at the end of the synthesis. Theoligoribonucleotides modified in this way are thereby generallyprotected from hydrolysis by RNases but also from possible isomerizationreactions and can therefore be stored stable over long periods. The2'-hydroxyl protective group is generally then only eliminated shortlybefore use of the RNA.

The elimination from the support, and the cleavage of the amino andhydroxyl protective groups on the synthesized oligonucleotides werecarried out by generally known methods, for example as described by M.Gait (ed.):

Oligonucleotide Synthesis, a practical approach; IRL Press; Oxford 1984.

The examples which follow are intended to explain the invention further.The following abbreviations have been used:

Bz for benzoyl

Mthp for methoxytetrahydropyranyl

DansEOC for dansylethoxycarbonyl and

EA for ethyl acetate

EXAMPLE 1 Reaction of 2-dansylethanol with Trichloromethyl Chloroformate

0.8 ml (1.32 g=6.64 mmol) of trichloromethyl chloroformate is pipettedinto 10 ml of absolute CH₃ CN while cooling in ice and stirring. Then,while cooling in ice and stirring, 1 g (3.58 mmol) of 2-dansylethanoldissolved in 5 ml of absolute CH₃ CN is added dropwise with a syringethrough a septum. The mixture is stirred in an ice bath for a further 5h. The precipitated colorless solid is filtered off with suction, washedwith absolute tetrahydrofuran and dried under high vacuum. 1.135 g (3.00mmol=84%) of a colorless solid of melting point 154-55° are obtained.

Elemental analysis shows:

    ______________________________________                                                      Found Calculated                                                ______________________________________                                        Carbon          47.90%  47.63%                                                Hydrogen        4.64%   4.53%                                                 Nitrogen        3.96%   3.70%                                                 ______________________________________                                    

EXAMPLE 2 Reaction of 2'-O-(4-methoxytetrahydropyranyl)-N⁶-benzoyl-adenosine with 2-dansylethyl Chloroformate Hydrochloride

2.43 g (5 mmol) of 2'-O-Mthp-N⁶ -Bz-adenosine are coevaporated 2× with30 ml of absolute pyridine each time and then dissolved in 40 ml ofabsolute pyridine. Then, while cooling in ice and stirring, 2.46 g (6.5mmol=1.3 eq) 2-dansylethyl chloroformate hydrochloride are added insolid form. The mixture is stirred in an ice bath for 1 h, thehydrochloride dissolving after about 0.5 h. The reaction is then stoppedwith 0.5 ml (8.8 mmol) of glycol, the mixture is concentrated in arotary evaporator, diluted with 200 ml of CH₂ Cl₂ and washed with 200 mlof saturated NaHCO₃ solution, the aqueous phase is extracted 2× with 100ml of CH₂ Cl₂ each time, and the combined organic phases are dried withNa₂ SO₄, filtered and concentrated in a rotary evaporator. They arecoevaporated 2× with 100 ml of toluene each time and 2× with 100 ml CH₂Cl₂ each time. Purification is carried out on an SiO₂ column (100 g,23×3.5 cm). Flash chromatography elution is carried out with 0.5 l ofCH₂ Cl₂, 1 l of CH₂ Cl₂ /MeOH 100:1 and 1.5 l of CH₂ Cl₂ /MeOH 100:2.The individual product fractions are concentrated in a rotary evaporatorand dried under high vacuum. 2.86 g (3.62 mmol=72%) of 5'-substitutedand 0.47 g (0.43 mmol=9%) of 3',5'-disubstituted product are obtained,in each case as yellow, highly fluorescent foams. For the elementalanalysis 100 mg of the disubstituted product is purified again on 1 SiO₂plate (40×20 cm) with CH₂ Cl₂ /MeOH 100:2.

Analysis data for2'-O-(4-methoxytetrahydropyranyl)-5'-O-dansylethoxycarbonyl-N⁶-benzoyladenosine

a) Thin-layer chromatography

The thin-layer chromatography was carried out on Schleicher und Schullsilica gel F1500/LS 254 in CH₂ Cl₂ /MeOH (95:5), and an R_(f) of 0.48was calculated.

b) UV spectroscopy in methanol:

    ______________________________________                                        +λmax                                                                            257           272    339                                            logε.sub.max                                                                    4.42          4.38   3.65                                           ______________________________________                                    

c) Elemental analysis

    ______________________________________                                                           Calculated with                                                         Found 1 mole of H.sub.2 O                                        ______________________________________                                        Carbon         56.58%  56.43%                                                 Hydrogen       5.46%   5.48%                                                  Nitrogen       10.28%  10.39%                                                 ______________________________________                                    

d) NMR spectroscopy

in CDCl₃ at 250 MHz

9.03 s broad (1) NH, 8.81 s (1) H-8, 8.61 d (2) dansyl H-2, 8.35-8.30 m(2) dansyl H-4, dansyl H-8, 8.23 s (1) H-2, 8.03 d (2) 2H of o-Bz,7.65-7.50 mm (5) 3H of Bz, dansyl H-3, dansyl H-7, 7.20 d (1) dansylB-6, 6.20 d (1) H-1', 5.13 t (1) B-2', 4.52-4.32 m (6) CH₂ OCO, B-3',H-4', H-5', H-5", 3.72 t (2) SO₂ CH₂, 3.77-3.43 m (4) CH₂ OCH₂ (Mthp),about 2.90 s broad (1) 3'-OH, 2.89 s (6) NMe₂, 2.87 s (3) OCH₃,1.95-1.55 m (4) CH₂ CCH₂ (Mthp)

Analysis data for2'-O-(4-methoxytetrahydropyranyl)-3',5'-bis-O-dansylethoxycarbonyl-N⁶-benzoyladenosine

a) Thin-layer chromatography

The thin-layer chromatography was carried out on silica gel F1500/LS 254(Schleicher & Schull) in CH₂ Cl₂ /MeOH (100:1), and an R_(f) of 0.35 wascalculated.

b) UV spectroscopy in methanol:

    ______________________________________                                        λmax                                                                             254           278    343                                            logε.sub.max                                                                    4.58          4.37   3.91                                           ______________________________________                                    

c) Elemental analysis

    ______________________________________                                                      Found Calculated                                                ______________________________________                                        Carbon          57.87%  58.07%                                                Hydrogen        5.46%   5.24%                                                 Nitrogen        8.67%   8.94%                                                 ______________________________________                                    

d) NMR spectroscopy

in CDCl₃ at 250 MHz

9.05 s broad (1) NH, 8.83 s (1) H-8, 8.69-8.60 2d (2) 2×dansyl H-2,8.35-8.29 m (4) 2×dansyl H-4, 2×dansyl H-8, 8.22 s (1) H-2, 8.03 d (2)2H of o-Bz, 7.68-7.50 m (7) 3H of Bz, 2×dansyl H-3, 2×dansyl H-7,7.24-7.18 2 d superimposed to give t (2) 2×dansyl H-6, 6.13 d (1) H-1',5.32 t (1) H-2', 5.13-5.11 m (1) H-3', 4.60-4.22 m (7) 2×CH₂ OCO, H-4',H-5', H-5", 3.77-3.26 m (8) 2×SO₂ CH₂, CH₂ OCH₂ (Mthp), 2.89 is (6)NMe₂, 2.88 s (6) NMe₂, 2.63 s (3) OCH₃, 1.78-1.20 m (4) CH₂ CCH₂ (Mthp)

EXAMPLE 3 Reaction of2'-O-(4-methoxytetrahydropyranyl)-5'-O-dansylethoxycarbonyl-N⁶-benzoyladenosine with 2-(4-nitrophenyl)ethyldiisopropylphosphoramidochloridite

1 g (1.264 mmol) of 2'-O-Mthp-5'-O-dansEOC-N⁶ -Bz-adenosine is dissolvedin 6 ml of absolute CH₂ Cl₂, and then 0.86 ml (0.65 g=5.03 mmol=4 eq) ofHunig's base and 0.84 g (2.528 mmol=2 eq) of 2-(4-nitrophenyl)ethyldiisopropylphosphoramidochloridite are added. The mixture is stirred atroom temperature under a nitrogen atmosphere and wrapped in aluminumfoil to exclude light. After 11/4 h, another 0.42 g (1.264 mmol=1 eq) ofphosphitylation reagent is added. After stirring at room temperature fora total of 2.5 h, the mixture is diluted with 75 ml of CH₂ Cl₂ andwashed with 75 ml of saturated NaHCO₃ solution, the aqueous phase isback-extracted 4× with 50 ml of CH₂ Cl₂ each time, and the combinedorganic phases are dried over Na₂ SO₄, filtered and concentrated in arotary evaporator. Purification is by flash chromatography on an SiO₂column (30 g, 12×3 cm), elution with 250 ml of CH₂ Cl₂, 100 ml of CH₂Cl₂ /EA 100:1, 100 ml of CH₂ Cl₂ /EA 100:2, 100 ml of CH₂ Cl₂ /EA 100:3,100 ml of CH₂ Cl₂ /EA 100:5, 100 ml of CH₂ Cl₂ /EA 100:7, 100 ml of CH₂Cl₂ /EA 9:1, 350 ml of CH₂ Cl₂ /EA 4:1 (reagent), 100 ml of CH₂ Cl₂ /EA2:1 (product), 100 ml of CH₂ Cl₂ /EA 1:1 (product), 100 ml of CH₂ Cl₂/EA 1:2 (product) and 100 ml EA (product).

The product fractions are concentrated in a rotary evaporator and driedunder high vacuum. 0.955 g (0.878 mmol=70%) of a yellow fluorescent foamis obtained.

Analytical data

a) Thin-layer chromatography

The thin-layer chromatography was carried out on Schleicher und Schullsilica gel F1500/LS 254 in toluene/EA (1:6), and an R_(f) of 0.50 wascalculated.

b) UV spectroscopy in methanol

    ______________________________________                                        λmax                                                                             261           330    339                                            logε.sub.max                                                                    4.54          3.55   3.51                                           ______________________________________                                    

c) Elemental analysis

    ______________________________________                                                      Found Calculated                                                ______________________________________                                        Carbon          57.08%  57.45%                                                Hydrogen        5.87%   5.84%                                                 Nitrogen        10.29%  10.31%                                                ______________________________________                                    

d) NMR spectroscopy

1. ³¹ P-NMR in CDCl₃ at 161.70 MHz

151.34 ppm s (31%)

149.47 ppm s (69%)

2. ¹ H-NMR in CDCl₃ at 250 MHz

9.07 s broad (1) NH, 8.83 and 8.82 2s (1) H-8, 8.61 d (1) dansyl H-2,8.35-8.29 m (2) dansyl H-4, dansyl H-8, 8.24 and 8.22 2s (1) H-2,8.18-8.13 2d (2) 2Ho to phenyl-NO₂, 8.03 d (2) 2H from o-Bz, 7.64-7.49 m(5) 3H of Bz, dansyl H-3, dansyl H-7, 7.45-7.38 2d (2) 2H m tophenyl-NO₂, 7.19 d (1) dansyl H-6, 6.23 and 6.15 2d (1) H-1', 5.20 and5.11 2t (1) H-2', 4.48 t (2) CH₂ OCO, 4.44-4.18 m (4) H-3', H-4', H-5',H-5", 4.10-3.88 m (2) CH₂ OP, 3.84-3.25 m (6) 2×CH (i-Pr). CH₂ OCH₂(Mthp), 3.71 t (2) SO₂ CH₂, 3.06 t (2) CH₂ -phenyl-NO₂, 2.88 s (6) NMe₂,2.66 and 2.61 2s (3) OCH₃, 2.00-1.45 m (4) CH₂ CCH₂ (Mthp, 1.26-1.12superimposed d (12) 2×C(CH₃)₂ (i-Pr)

EXAMPLE 4 Automatic Oligoribonucleotide Synthesis with2'-O-(4-methoxytetrahydropyranyl)-5'-O-dansylethoxycarbonylphosphoramidites. Preparation of the decanucleotide (rAp)₉ T

The syntheses were carried out with a 380 B DNA synthesizer (AppliedBiosystems)

Column used: ABI standard column

Support material used: LCAMA-CPG support which is linked to thenucleoside via the 3'-hydroxyl group.

(Reference: K. P. Stengele, W. Pfleiderer Nucleic Acids Res. Symp. Ser.21, 101 (1989); K. P. Stengele, W. Pfleiderer Tetrahedron Lett. 31, 2549(1990)).

Loading with thymidine which is linked via the 3'-hydroxyl group to thesupport; 19 μmol/g

Batch size: about 0.6 μmol (determination by trityl elimination).

Synthesis cycle:

1. Condensation with 0.5 M tetrazole and 0.1 M2'-O-(4-methoxytetrahydropyranyl)-5'-O-dansylethoxycarbonyl-N⁵-benzoyladenosine 3'-O-phosphoramidite in absolute acetonitrile inaccordance with the following pulse sequence:

    ______________________________________                                                              tetrazole                                                                              8      sec.                                    Phosphoramidite                                                                           +         tetrazole                                                                              4      sec.                                                          tetrazole                                                                              3      sec.                                    Phosphoramidite                                                                           +         tetrazole                                                                              3      sec.                                                          tetrazole                                                                              3      sec.                                    Waiting step                   60     sec.                                    Phosphoramidite                                                                           +         tetrazole                                                                              3      sec.                                                          tetrazole                                                                              3      sec.                                    Waiting step                   700    sec.                                    ______________________________________                                    

2. Capping of unreacted nucleotide with acetic anhydride/lutidine/THF(1:1:3) and 6.5% dimethylaminopyridine (DMAP) in THF

    ______________________________________                                               Flow-through                                                                           20 sec.                                                              Waiting step                                                                           30 sec.                                                       ______________________________________                                    

3. Oxidation with I₂ solution (1.269 g I₂ /20 ml H₂ O/10 ml pyridine/100ml THF)

    ______________________________________                                               Flow-through                                                                           30 sec.                                                              Waiting step                                                                           30 sec.                                                       ______________________________________                                    

4. Dansylethoxycarbonyl elimination with 0.1 M DBU in acetonitrile in2×30 sec. and 8×10 sec. pulsed flows with interpolated 1 sec reverseflushes.

The eluates from the 4th step were collected and the condensation yieldswere determined on the basis of the 5-dimethylamino-1-naphthyl vinylsulfone which was formed by means of fluorescence spectroscopy(excitation: 368 nm; emission: 526 nm).

The average stepwise yield was about 98%.

Between the individual steps 1-4 the customary washing steps withacetonitrile and the block and reverse flushes were carried out.

EXAMPLE 5 Synthesis of 5'-O-dansylethoxycarbonyl-protectedH-phosphonates

10.75 equivalents of imidazole are dissolved in 5 ml of absolutemethylene chloride and then cooled with ice/sodium chloride, andsubsequently 3.5 equivalents of PCl₃ and 11.25 equivalents oftriethylamine are added to the cooled solution. The mixture is stirredwhile cooling for 15 minutes and then 0.25 mmol (1 equivalent) of2'-O-(4-methoxytetrahydropyranyl)-5'-O-dansylethoxycarbonyl-N⁶-benzoyladenosine or 0.25 mmol of2'-O-(4-methoxytetrahydropyranyl)-5'-O-dansylethoxycarbonyl-N⁶-para-nitrophenylethyloxycarbonyladenosine (coevaporated 1× withacetonitrile) in 5 ml of absolute methylene chloride is added dropwisewith stirring over the course of 10 min.

The ice bath is then removed and the mixture is stirred at roomtemperature for a further 15 minutes. The reaction solution issubsequently extracted by shaking with 10 ml of 1 M triethylammoniumbicarbonate. The phases are separated, the aqueous phase is extractedwith 10 ml of CH₂ Cl₂, and the combined organic phases are dried overNa₂ SO₄, filtered and concentrated in a rotary evaporator to a yellowfluorescent foam. It is purified on a short silica gel column (flashchromatography) with a CH₂ Cl₂ /MeOH gradient.

EXAMPLE 6 Automatic Oligoribonucleotide Synthesis with2'-O-(4-methoxytetrahydropyranyl)-5'-O-dansylethoxycarbonyl-ribonucleotide3'-O-H-phosphonates. Preparation of (rAp)₉ T

The syntheses were carried out with a 380 B DNA synthesizer (AppliedBiosystems).

Column used: ABI standard column

Support material used: LCAMA-CPG support (reference: K. P. Stengele, W.Pfleiderer Nucleic Acids Res. Symp. Ser. 21, 101 (1989); K. P. Stengele,W. Pfleiderer Tetrahedron Lett. 31, 2549 (1990)).

Loading with thymidine which is linked via the 3'-hydroxyl group to thesupport, 19 μmol/g

Batch size: about 0.6 μmol (determination by trityl elimination).

Synthesis cycle:

1. Washing with absolute pyridine/acetonitrile (1:1)

2. Reaction with2'-O-(4-methoxytetrahydropyranyl)-51-O-dansylethoxycarbonylH-phosphonate (10 mM) and pivaloyl chloride (50 mM) in absolutepyridine/acetonitrile (1:1)

3. Washing with absolute acetonitrile (45 seconds)

4. Dansylethoxycarbonyl elimination with 0.1 M DBU in acetonitrile (2minutes)

5. Repetition of steps 1 to 4 until the required chain length isreached.

6. Dansylethoxycarbonyl elimination with 0.1 M DBU in acetonitrile (2minutes) and collection of the eluates

7. Oxidation with I₂ (0.1 M) in pyridine/N-methylimidazole/water/THF(5/1/5/90) (2.5 minutes) or with I₂ (0.1 M) in triethylamine/water/THF(5/5/90) (2.5 minutes)

The oxidation to the thiophosphate or phosphoramidate was also carriedout as described in Uhlmann & Peyman (1990).

The eluates from the 6th step are collected and the condensation yieldsare determined on the basis of the 5-dimethylamino-1-naphthyl vinylsulfone which is formed by means of fluorescent spectroscopy(excitation: 368 nm; emission: 526 nm).

The average stepwise yield is about 98%.

We claim:
 1. A compound of the formula (Va) or (Vb) ##STR24## in which:(i) DansEOC is a group of the formula ##STR25## (ii) R₁ is selected fromthe group consisting of ##STR26## (iii) B is selected from the groupconsisting of ##STR27## wherein (A) R₂ is selected from the groupconsisting of ##STR28## (B) R₃ is selected from the group consisting of##STR29## (C) R₄ is selected from the group consisting of ##STR30## (D)R₅ is selected from the group consisting of ##STR31## (E) Y is selectedfrom the group consisting of --H, --CH₃, and n-alkyl C₁ -C₄ ;(iv) R₆ andR₇ are the same or different and are selected from the group consistingof: a C₁ -C₈ -alkyl group, a C₅ -C₁₂ -cycloalkyl group, a benzyl group,and a phenyl group, or, alternatively, R₆ and R₇, together with thenitrogen atom to which they are bonded, form a substituted orunsubstituted, saturated or unsaturated, heterocyclic ring; and (v) R₈is selected from the group consisting of ##STR32## an unsubstitutedbenzyl group, and a ring-substituted benzyl group, where thesubstituent(s), independently of one another, are selected from thegroup consisting of a halogen, a C₁ -C₄ -alkyl group, nitro, methoxy,and a carboxyl group.
 2. A process for the preparation of a compound ofthe formula (Va) or (Vb) ##STR33## which comprises (a) reacting acompound of the formula (IIIa) or (IIIb) ##STR34## in which: (i) DansEOCis a group of the formula ##STR35## (ii) R₁ is selected from the groupconsisting of ##STR36## (iii) B is selected from the group consisting of##STR37## wherein (A) R₂ is selected from the group consisting of##STR38## (B) R₃ is selected from the group consisting of ##STR39## (C)R₄ is selected from the group consisting of ##STR40## (D) R₅ is selectedfrom the group consisting of ##STR41## (E) Y is selected from the groupconsisting of --H, --CH₃, and n-alkyl C₁ -C₄ ;(b) in the presence of abase with a compound of the formula (VI) ##STR42## in which: (i) R₆ andR₇ are the same or different and are selected from the group consistingof C₁ -C₈ -alkyl, C₅ -C₁₂ cycloalkyl, benzyl, and phenyl, or,alternatively, R₆ and R₇, together with the nitrogen atom to which theyare bonded, form a substituted or unsubstituted, saturated orunsaturated heterocyclic ring, (ii) R₈ is selected from the groupconsisting of ##STR43## an unsubstituted benzyl group, and aring-substituted benzyl group, where the substituent(s) are selectedfrom the group consisting of a halogen, a C₁ -C₄ -alkyl group, nitro,methoxy, and a carboxyl group, and (iii) Z is selected from the groupconsisting of chlorine, bromine, and a radical of the formula --NR₉ R₁₀,where R₉ and R₁₀ are the same or different and are selected from thegroup consisting of C₁ -C₈ -alkyl, C₅ -C₁₂ cycloalkyl, benzyl group, andphenyl.
 3. In a process for the preparation of oligonucleotides, theimprovement which comprises:reacting a compound of the formula (Va) or(Vb) ##STR44## in which: (i) DansEOC is a group of the formula ##STR45##(ii) R₁ is selected from the group consisting of ##STR46## (iii) B isselected from the group consisting of ##STR47## wherein (A) R₂ isselected from the group consisting of ##STR48## (B) R₃ is selected fromthe group consisting of ##STR49## (C) R₄ is selected from the groupconsisting of ##STR50## (D) R₅ is selected from the group consisting of##STR51## (E) Y is selected from the group consisting of --H, --CH₃, andn-alkyl C₁ -C₄ ; (iv) R₆ and R₇ are the same or different and areselected from the group consisting of: a C₁ -C₈ -alkyl group, a C₅ -C₁₂-cycloalkyl group, a benzyl group, and a phenyl group, or,alternatively, R₆ and R₇, together with the nitrogen atom to which theyare bonded, form a substituted or unsubstituted, saturated orunsaturated, heterocyclic ring; and (v) R₈ is selected from the groupconsisting of ##STR52## an unsubstituted benzyl group, and aring-substituted benzyl group, where the substituent(s), independentlyof one another, are selected from the group consisting of a halogen, aC₁ -C₄ -alkyl group, nitro, methoxy, and a carboxyl group; to form anoligonucleotide comprising said compound of the formula (Va) or (Vb). 4.A compound of the formula (VIIIa) or (VIIIb) ##STR53## in which: (i)DansEOC is a group of the formula ##STR54## (ii) R₁ is selected from thegroup consisting of ##STR55## (iii) B is selected from the groupconsisting of ##STR56## wherein (A) R₂ is selected from the groupconsisting of ##STR57## (B) R₃ is selected from the group consisting of##STR58## (C) R₄ is selected from the group consisting of ##STR59## (D)R₅ is selected from the group consisting of ##STR60## (E) Y is selectedfrom the group consisting of --H, --CH₃, and n-alkyl C₁ -C₄ ; and(iv)K.sup.(+) is a cation.
 5. A process for the preparation of a compound ofthe formula (VIIIa) or (VIIIb) ##STR61## which comprises: (a) reacting acompound of the formula (IIIa) or (IIIb) ##STR62## in which: (i) DansEOCis a group of the formula ##STR63## (ii) R₁ is selected from the groupconsisting of ##STR64## (iii) B is selected from the group consisting of##STR65## wherein (A) R₂ is selected from the group consisting of##STR66## (B) R₃ is selected from the group consisting of ##STR67## (C)R₄ is selected from the group consisting of ##STR68## (D) R₅ is selectedfrom the group consisting of ##STR69## (E) Y is selected from the groupconsisting of --H, --CH₃, and n-alkyl C₁ -C₄ ;(iv) and where K⁺ is acation; (b) in the presence of a base, with a compound of the formula(IX)

    PR.sub.14 R.sub.15 R.sub.16                                (IX)

in which R₁, R₁₅, and R₁₆ are the same or different and are selectedfrom the group consisting of hydrogen, a C₁ -C₈ -alkyl group, a C₁ -C₈-fluoroalkyl group, and an aryl group.
 6. A process for the preparationof a compound of the formula (VIIIa) or (VIIIb) ##STR70## whichcomprises: (a) reacting a compound of the formula (IIIa) or (IIIb)##STR71## in which: (i) DansEOC is a group of the formula ##STR72## (ii)R₁ is selected from the group consisting of ##STR73## (iii) B isselected from the group consisting of ##STR74## wherein (A) R₂ isselected from the group consisting of ##STR75## (B) R₃ is selected fromthe group consisting of ##STR76## (C) R₄ is selected from the groupconsisting of ##STR77## (D) R₅ is selected from the group consisting of##STR78## (E) Y is selected from the group consisting of --H, --CH₃, andn-alkyl C₁ -C₄ ;(iv) and where K⁺ is a cation; (b) in the presence of abase, with a compound of the formula (X)

    PR.sub.17 R.sub.18 R.sub.19                                (X)

in which R₁₇, R₁₈, and R₁₉ are the same or different and are selectedfrom the group consisting of chlorine, bromine, a C₁ -C₈ -alkylaminogroup, and a 1,2,4-triazolyl group; and (c) hydrolyzing the resultingproduct.
 7. In a process for protecting a hydroxyl group duringoligonucleotide synthesis, the improvement which comprisesreacting acompound of the formula (I) ##STR79## with said hydroxyl group such thatsaid hydroxyl group is protected during oligonucleotide synthesis.
 8. Ina process for the synthesis of oligonucleotides, the improvement whichcomprises reactinga compound of formula (IIIa) or (IIIb) ##STR80## inwhich: (i) DansEOC is a group of the formula ##STR81## (ii) R₁ isselected from the group consisting of: ##STR82## (iii) B is selectedfrom the group consisting of: ##STR83## wherein (A) R₂ is selected fromthe group consisting of: ##STR84## (B) R₃ is selected from the groupconsisting of: ##STR85## (C) R₄ is selected from the group consisting of##STR86## (D) R₅ is selected from the group consisting of ##STR87## (E)Y is selected from the group consisting of --H, --CH₃, and n-alkyl C₁-C₄, to form an oligonucleotide comprising said compound of the formula(IIIa) or (IIIb).
 9. A compound of the formula (Va) or (Vb) as claimedin claim 1, in which R₆ and R₇ are the same or different and areselected from the group consisting of an isopropyl group and a C₅ -C₈cycloalkyl group.
 10. A process as claimed in claim 2, in which R₆ andR₇, independently of one another, are selected from the group consistingof an isopropyl group and a C₅ -C₈ cycloalkyl group.
 11. A process asclaimed in claim 2 in which Z is chlorine.
 12. A process as claimed inclaim 2, in which said base comprises(a) a pyridine, or (b) a mixture oftetrahydrofuran, dioxane, methylene chloride, chloroform, and/oracetonitrile with(i) a C₁ -C₄ -trialkylamine or (ii) tetrazole.
 13. Aprocess as claimed in claim 12, in which said C₁ -C₄ -trialkylamine isselected from the group consisting of trimethylamine, triethylamine, ordiisopropylethylamine.
 14. A process as claimed in claim 12, in which Zis a radical of the formula --NR₉ R₁₀ and said C₁ -C₄ -trialkylamine hasthe formula HNR¹¹ R¹² R¹³(+) X.sup.(-), where one or more of R¹¹, R₁₂and R₁₃ are the same or different and are a C₁ -C₄ -alkyl group, and Xis a halogen.
 15. A process as claimed in claim 14, in which X ischlorine.
 16. A process as claimed in claim 12, in which said basecomprises a mixture of tetrahydrofuran, dioxane, methylene chloride,chloroform, and/or acetonitrile with tetrazole.
 17. A process for thepreparation of oligonucleotides, which comprises:(a) reacting a compoundof the formula (Va) or (Vb) as claimed in claim 1 (b) with a compound ofthe formula (VIIa) or (VIIb) ##STR88## wherein G has the same meaning asR₁ or is a polymeric support which is bonded via the 2'-hydroxyl or3'-hydroxyl group of said compound of the formula (VIIa) or (VIIb), (c)oxidizing the resulting compound, (d) eliminating thedansylethoxycarbonyl group, (e) reacting the resulting compound with acompound of the formula (Va) or (Vb) as claimed in claim 1, and (f)repeating steps (c), (d), and (e) until the desired chain length isobtained.
 18. A process as claimed in claim 17, in which said compoundof the formula (Va) or (Vb) is reacted with said compound of the formula(VIIa) or (VIIb) at a temperature of -20 to +100° C.
 19. A process asclaimed in claim 18, in which said temperature is room temperature. 20.A process as claimed in claim 17, wherein the reaction is carried out inthe presence of a weak acid.
 21. A process as claimed in claim 17,wherein the reaction is carried out in the presence of tetrazole orp-nitrophenyltetrazole.
 22. A process as claimed in claim 17, whereinthe oxidation is carried out with iodine, sulfur, or iodine in thepresence of an amine.
 23. A process as claimed in claim 17, wherein theoxidation is carried out at a temperature of -80 to +100° C.
 24. Aprocess as claimed in claim 23 wherein said temperature is -20 to +60°C.
 25. A process as claimed in claim 23, wherein said temperature isroom temperature.
 26. A dansylethoxycarbonyl-protected oligonucleotidecomprising at least one compound of the formula (Va) or (Vb) as claimedin claim
 1. 27. An oligonucleotide prepared from at least one compoundof the formula (Va) or (Vb) as claimed in claim
 1. 28. A compound asclaimed in claim 4, in which K.sup.(+) is HN(C₂ H₅)_(3!).sup.(+).
 29. Aprocess as claimed in claim 5, in which R₁₄, R₁₅, and R₁₆ can be thesame or different and are selected from the group consisting of a2,2,2-trifluoroethyl group, a 1,1,1,3,3,3,-hexafluoro-2-propyl group, anethyl group, and a phenyl group.
 30. A process as claimed in claim 6, inwhich one or more of R₁₇, R₁₈, and R₁₉, is a 1,2,4-triazolyl group. 31.A process as claimed in claim 6, wherein said base is C₁ -C₄-trialkylamine or N-C₁ -C₄ -alkylmorpholine.
 32. A process as claimed inclaim 31, wherein said base is N-methylmorpholine.
 33. In a process forthe preparation of oligonucleotides, the improvement whichcomprisesreacting a compound of the formula (VIIIa) or (VIIIb) asclaimed in claim 6 to form an oligonucleotide comprising said compoundof the formula (VIIIa) or (VIIIb).
 34. A process for the preparation ofoligonucleotides, which comprises:(a) reacting a compound of the formula(VIIIa) or (VIIIb) as claimed in claim 6, (b) with a compound of theformula (VIIa) or (VIIb), ##STR89## where G has the same meaning as R₁or is a polymeric support which is bonded via the 2'-hydroxyl or3'-hydroxyl group of said compound of the formula (VIIa) or (VIIb), (c)eliminating the dansylethoxycarbonyl group, (d) reacting the resultingcompound with a compound of the formula (VIIIa) or (VIIIb), (e)repeating steps (c) and (d) until the desired chain length is obtained,and (f) oxidizing the resulting oligonucleotide.
 35. A process asclaimed in claim 34, wherein the compound of the formula (VIIIa) or(VIIIb) is reacted with the compound of the formula (VIIa) or (VIIb) ata temperature of -20 to +100° C.
 36. A process as claimed in claim 35,wherein said temperature is room temperature.
 37. A process as claimedin claim 34, wherein the reaction is carried out in the presence of anacid halide.
 38. A process as claimed in claim 37, wherein said acidhalide is an acid chloride.
 39. A process as claimed in claim 34,wherein the reaction is carried out in the presence of pivaloyl chlorideor adamantanecarbonyl chloride.
 40. A process as claimed in claims 34,wherein the oxidation is carried out with iodine, sulfur, or an amine inthe presence of triphenylphosphine/CCl₄.
 41. Adansylethoxycarbonyl-protected oligonucleotide prepared from at leastone compound of the formula (VIIIa) or (VIIIb) as claimed in claim 6.42. An oligonucleotide prepared from at least one compound of theformula (VIIIa) or (VIIIb) as claimed in claim 6.